Fig 2: The CRBN65 antibody detects CRBN protein across a wide range of MM cell lines with high specificity. A panel of MM cell extracts (20 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000) or ß-tubulin, without blocking peptide (upper panel) or with blocking peptide (lower panel). CRBN, cereblon; kDa, kiloDalton; MM, multiple myeloma; rCRBN, recombinant cereblon.

Fig 3: The CRBN65 antibody detects cereblon (CRBN) protein with high specificity compared with commercial antibodies. DF15 and DF15R MM cell extracts (20 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 (1:1000) or commercial antibodies (manufacturers' recommended dilution) against CRBN. Blots were analysed on LI-COR Oddysey and arrows denote the full-length CRBN protein band [51 kiloDalton (kDa)] that appears specifically in DF15 cells but not DF15R.

Fig 4: A complex profile of CRBN splice variants exists in both MM cell lines and primary CD138+ MM tumour cells. (A) Nested polymerase chain reaction (PCR) reaction for CRBN splice variants in H929 MM cells were separated on an agarose gel and arrows indicate those bands that were gel purified and sequenced. (B) Nested PCR reaction for CRBN splice variants in 8 MM patient (pt) cells were separated on an agarose gel and multiple bands indicate complexity of CRBN mRNA profile. CRBN, cereblon; MM, multiple myeloma.

Fig 5: The CRBN65 antibody detects recombinant and endogenous CRBN protein with high sensitivity. (A) Recombinant human full-length CRBN (0·2–60 ng) along with DF15 and DF15R cell extracts (20 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000). (B) Linear regression analysis using CRBN signal as measured on LI-COR Oddysey for rhCRBN is linear in the 0·2–60 ng range, and CRBN protein measured in DF15 cells falls within linear range of detection. CRBN, cereblon; kDa, kiloDalton; Mol W, molecular weight; R2, coefficient of determination.